Determination of adenosine phosphates in mouse myocardium tissue by HPLC with UV detection and using porous graphite carbon column

Abstract

A high-performance liquid chromatography (HPLC) method with UV detection was established and validated for the simultaneous determination of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in mouse myocardial tissues. After protein precipitation and compound extraction with pre-cooled perchloric acid and the supernatant was centrifuged with the pH value adjusted to 6.5–7.5, the analytes were separated on a porous graphitic carbon LC column (4.6 mm × 100 mm, 5 μm) using gradient elution with a mobile phase of 10 mmol/L borax solution, pH 9.18(A) and acetonitrile-tetrahydrofuran (1:1, v/v) (B). The LC flow rate was 0.8 mL/min; the UV detection wavelength was 254 nm and the column temperature was maintained at 35 °C. ATP, ADP, and AMP were separated and the intra-day relative standard deviations (RSDs) of peak area repeatability were 1.3–2.5% (n = 6). The correlation coefficients of the linearity between UV responses and adenosine phosphate concentrations were larger than 0.9998 in all cases, within concentration ranges of 0.71–91.6 μg/mL for ATP, 1.3–81.5 μg/mL for ADP and 1.69–108.1 μg/mL for AMP. The limits of detection were within 0.17–0.21 μg/mL. The average standard substance spiked-in recoveries were 93.6–104.7% (n = 3). The established HPLC method was successfully applied to quantitate ATP, ADP, and AMP in mouse myocardial tissues.